CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping

Abstract

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively-multiplexed yeast two-hybrid method, CrY2H-seq, that uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins, and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated combinatorial interactions among plant transcription factors. By performing ten independent CrY2H-seq screens each testing 3.6 million interaction combinations, and reporting a deep coverage network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq’s improved capacity, efficiency, and sensitivity over existing technologies. In addition to recapitulating one third of previously reported interactions derived from diverse methods, we expand the number of reported plant transcription factor interactions by three-fold, revealing previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.

Systematic Dissection of Sequence Elements Controlling σ70 Promoters Using a Genomically-Encoded Multiplexed Reporter Assay inE. coli

“…Though we integrated over 300,000 unique promoter barcode combinations, we expect the methods are easily scalable and compatible with interrogating millions of variants. Furthermore, this approach should work for any of the many model systems in which Cre-recombination is available [57][58][59] and for a wide variety of reporter assay formats, especially those which require single variants per cell. While the methods we use to generate landing pads and perform the subsequent cassette exchange have been previously established, to our knowledge we are the first to use them in this manner.…”

Exploring the protein–protein interaction landscape in plants

“…Thus, CrY2H‐seq offers a cost‐effective and time‐efficient alternative for the classical Y2H method with improved screening capacity, efficiency, and sensitivity. CrY2H‐seq is a promising approach that can be further optimized, for instance, for different variants of the Y2H assay, such as yeast one‐hybrid that allows screening of genome‐wide protein–DNA interactions or of a cDNA library against another cDNA library to compare large‐scale interactomes under different conditions or tissues (Trigg et al, ).…”

“…Despite all these disadvantages and the fact that Y2H is prone to yield relatively high false‐positive and false‐negative rates (Brückner et al, ), over the years, this system has proved to be efficient to discover many new PPIs. Y2H has been successfully used to create large interaction networks in many plant species, such as Arabidopsis thaliana (de Folter et al, ; Mukhtar et al, ; Trigg et al, ; Vernoux et al, ), barley ( Hordeum vulgare L.; Schoonheim et al, ), wheat ( Triticum aestivum ; Tardif et al, ), and rice ( Oryza sativa ; Ding et al, ). The classical Y2H system and all its variations have been extensively reviewed (Morsy et al ; Ferro & Trabalzini, ), including detailed technical descriptions, pitfalls, and benefits.…”

Removing auto-activators from yeast-two-hybrid assays by conditional negative selection

“…Recent approaches that combine next-generation sequencing (NGS) with Y2H are bringing us closer to obtaining genome-wide PPI networks (Weimann et al , 2013; Yachie et al , 2016). To avoid the manual, labor-intensive screening of bait proteins for tracking interactions, pools of baits are screened against pools of preys with multiplexed screening strategies (Yachie et al , 2016; Hastie and Pruitt, 2007; Trigg et al , 2017). Barcode Fusion Genetics (BFG-Y2H)(Yachie et al , 2016) utilizes intracellular DNA recombination of barcoded open reading frame (ORF) to identify interacting proteins, thus allowing pooling and simultaneous sequencing of Y2H-positive colonies.…”

“…Another approach, Cre-reporter-mediated Y2H coupled with next-generation sequencing (CrY2H-seq), uses Cre recombinase as a Y2H protein-protein interaction reporter. Cre covalently and unidirectionally links the interacting bait and prey plasmids via specialized loxP sites that flank the protein-coding sequences (Trigg et al , 2017). The linked protein-coding sequences can be amplified by PCR and serve as interaction-identifying DNA molecules that can be sequenced by NGS approaches, thus allowing massively multiplexed screening for protein–protein interactions.…”